(A) Study pathogenesis and pathophysiology of high biocontainment viral pathogens utilizing molecular technologies including reverse genetics systems: The past year we developed a model for Crimean-Congo hemorrhagic fever (CCHF) infection in type I interferon deficient mice using the clinical CCHFV isolate strain Hoti. Mice infected with strain Hoti develop a progressively worsening and ultimately fatal disease. We further developed the first CCHFV nonhuman primate disease model. Cynomolgus macaques exhibited hallmark signs of human CCHF with remarkably similar viral dissemination, organ pathology and disease progression. Histopathology showed infection of hepatocytes, endothelial cells and monocytes and fatal outcome seemed associated with endothelial dysfunction manifesting in a clinical shock syndrome with coagulopathy. This non-human primate model will be an invaluable asset for CCHFV countermeasures development. (Haddock et al. Nature Microbiol 2018) We further studied the pathogenicity of multiple Ebola virus, strain Makona, isolates. The study was initiated after in vitro reports that Makona accumulated mutations in the glycoprotein gene that were associated with adaptation to humans during the West African epidemic. We tested different isolates displaying or lacking the questionable mutations utilizing a mouse and the rhesus macaque model. Surprisingly, all isolates behaved very similarly independent of the genotype, causing severe or lethal disease in mice and macaques, respectively. Likewise, we could not detect any evidence for differences in virus shedding. Thus, no specific biological phenotype could be associated with these EBOV-Makona mutations in vivo. (Haddock et al., J Infect Dis 2018; Marzi et al., Cell Rep 2018)) We have continued to utilize humanized mice to study ebolavirus pathogenesis and immune responses. To investigate differences in viral pathogenicity, humanized mice (hu-NSG-SGM3) were inoculated with Ebola virus (EBOV, causes fatal human disease) or Reston virus (RESTV, does not cause disease in humans). Consistent with differences in disease in human infection, pronounced weight loss and markers of hepatic damage and disease were observed exclusively in EBOV-infected mice. These abnormalities were associated with significantly higher EBOV replication in the liver but not in the spleen, suggesting that efficiency of viral replication in select tissues early in infection may contribute to differences in viral pathogenicity. (Lavender et al., j Infect Dis 2018; Spengler et al., J Infect Dis 2017) (B) Study immune responses to infection and vaccination of high containment viral pathogens and develop new vaccine candidates: The VSV vaccine efforts are reported under the 'Trivalent Filovirus Vaccine' project. In the meantime, we have proceeded with the development of alternative vaccine candidates. We report the construction and preclinical efficacy testing of a novel recombinant modified vaccinia Ankara (MVA)-based Ebola vaccine expressing the EBOV-Makona glycoprotein GP and matrix protein VP40 (MVA-EBOV). We report 100% protection against lethal Ebola infection in guinea pigs after prime/boost vaccination with MVA-EBOV. Furthermore, this vaccine protected macaques from lethal disease after a single dose or prime/boost vaccination. This is the first report that a replication-deficient MVA vector can confer full protection against lethal EBOV challenge after a single dose vaccination in macaques. (Domi et al., Sci Rep 2018) We report the development and assessment of the protective efficacy of an adenovirus (Ad)-based CCHFV vaccine expressing the nucleocapsid protein (N) of CCHFV (Ad-N) in a lethal immunocompromised mouse model of CCHF. The results show that Ad-N can protect mice from CCHF mortality and that this platform should be considered for future CCHFV vaccine strategies. (Zivcec et al., PLoS Negl Trop Dis 2018) (C) Study vector/reservoir transmission of high containment viral pathogens using appropriate animal models: We continued to study infection kinetics of different Lassa virus in the Mastomys reservoir utilizing a unique colony established here at RML. The animals support virus replication and shedding for several weeks before Lassa virus gets cleared. The model will allow for important transmission studies. (manuscript in preparation; studies ongoing) (D) Utilize in vitro and in vivo systems to study the interactions between viral pathogen or viral components and host cells and develop new antiviral strategies: Lassa virus, the cause of Lassa fever in humans, is endemic to West Africa. Treatment of Lassa fever is primarily supportive, although ribavirin has shown limited efficacy if administered early during infection. We tested favipiravir in Lassa virus-viremic macaques and found that 300 mg/kg daily for 2 weeks successfully treated infection. (Rosenke et al., Emerg Infect Dis 2018) We utilized the new mouse model to evaluate the efficacy of favipiravir against lethal challenge with CCHFV strains Hoti and 10200. While ribavirin treatment suppressed viral loads, it was unable to prevent development of terminal disease. In contrast, favipiravir showed clinical benefit even when administered late in the clinical progression of CCHF. (Hawman et al., Antiviral Res 2018) We have tested polyclonal and monoclonal antibodies for efficacy against filovirus infections in animal models. First, we utilized transchromosomal bovines (TcB) vaccinated with a DNA plasmid encoding Ebola virus glycoprotein sequence to produce human polyclonal antibodies directed against Ebola virus glycoprotein. When administered 1-day post infection, these TcB polyclonal antibodies provided partial protection and resulted in a 50% survival rate following a lethal challenge of Ebola virus in rhesus macaques. Next, we attempted to improve the therapeutic efficacy of the neutralizing mAb M4 by combining treatment with 1 or 2 of blocking but nonneutralizing mAbs 126-15 and 127-8. We found that single-dose treatment early after infection with the neutralizing mAb M4 or any of the mAb combinations resulted in similar protection in the MARV hamster model. However, a single-dose treatment with the cocktail of all 3 mAbs provided the best protection in delayed treatment, with 67%-100% of the animals surviving a lethal challenge depending on the time of treatment. (Rosenke et al., J Infect Dis 2018; Marzi et al., J Infect Dis 2018) (E) Study the epidemiology and ecology of high biocontainment pathogens utilizing newly developed rapid, sensitive and specific diagnostic test systems including those that can be applied under field conditions: Please see report on Mali ICER study. In response to the Zika virus (ZIKV) emergence we developed ZIKV mouse models for countermeasure development. We also developed two novel VSV-ZIKV vaccines utilizing the favorable immune targeting of the existing VSV-EBOV vector. In addition to the EBOV GP, these new vaccines express the full-length pre-membrane and envelope proteins or pre-membrane and truncated soluble envelope proteins as antigens. Efficacy testing in the mouse model resulted in uniform protection when a single dose was administered 28 days prior to lethal challenge. Furthermore, this vaccine is fast-acting and can uniformly protect mice from lethal disease when administered as late as 3 days prior to ZIKV challenge. (Marzi et al., Front Cell Infect Microbiol 2018; Emanuel et al., Sci Rep 2018)